The Orthopoxvirus genus of the family Poxviridae includes both the causative agent of smallpox, variola virus, and the vaccine strain used for smallpox eradication, Vaccinia virus. Even though naturally occurring smallpox has been successfully eradicated, we need to better understand the mechanisms supporting Orthopoxviruses dissemination in light of potential bioterrorist attacks and/or outbreaks of epizootic infections (monkeypox). The life cycle of Orthopoxviruses relies on the production of two infectious forms, the intracellular mature viruses (IMV) and the extracellular viruses (EV). EV is formed through wrapping of IMV in double membranes derived from the trans-Golgi network. The fusion of the outer membrane of EV to the plasma membrane leads to formation of cellular-associated EV (CEV). A central event in CEV dissemination is phosphorylation of the viral protein A36 by non-receptor tyrosine kinases of the Src family. A36 phosphorylation on tyrosine residues creates docking sites for the adaptor proteins Nck1 and Grb2. These adaptors mediate the recruitment of N- WASP, which mediates the recruitment and activation of the actin nucleator ARP2/3. In contrast to the molecular machinery leading to actin tail formation, the identity of the putative receptor tyrosine kinase mediating Src activation is unknown. Moreover, the identity of the viral protein that may engage this putative receptor is unknown as well. To address this gap in knowledge, we have combined our extensive expertise in the mechanisms supporting actin-based motility, high-throughput imaging and large-scale RNAi-based genetic investigation. In this R21 exploratory application, we propose (Aim1) to determine the receptor tyrosine kinase(s) involved in actin tail formation and (Aim2) to determine the viral protein(s) engaging receptor tyrosine kinase signaling at the plasma membrane. These exploratory studies may provide mechanistic insight into molecular and cellular processes relevant to the treatment and prevention of diseases caused by pathogens that exploit the actin cytoskeleton for their dissemination.